Acetyl esterase producing strains and methods of using same

ABSTRACT

Acetyl esterase producing bacterial strains or functional mutants thereof and methods of using acetyl esterase producing bacterial strains as forage additives are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/947,865, filed onSep. 23, 2004, which claims the benefit of U.S. Provisional ApplicationNo. 60/505,521, filed on Sep. 23, 2003, both of which are hereinincorporated by reference in their entirety.

TECHNICAL FIELD

Organisms that produce acetyl esterase and methods of using same toenhance plant dry matter and fiber digestion in animals, as well as,enhance the preservation of the ensiled forage are disclosed.

BACKGROUND OF INVENTION

The plant cell wall is a complex structure consisting of differentpolysaccharides, the major components being cellulose, hemicellulosesand pectins. The resistance of the plant cell wall to digestion presentssignificant challenges in the animal production industry. Presently, inlivestock agriculture while a high-forage diet is desirable, it does notcurrently satisfy the demands of modern animal production. Fiberdigestion is a limiting factor to dairy herd milk yield and composition,and to beef production in beef operations feeding a high forage diet,and hence restricts profitability of farmers. Enhancing fiber digestionhas a dual impact: 1) the animal eats more due to a reduced gut fill andtherefore produces more, and 2) the animal gets more out of what it eatssince the fiber is more digestible. Ultimately, this should increasemilk yield, in dairy cows, and beef production in forage fed animals.Farmers either have to put up with a lower level of feed digestibilityand hence productivity, or they can use inoculants, forage additives orother amendments that improve the digestibility of feed.

Accordingly, farmers can treat ensiled feed or other animal feed withfiber degrading enzymes, originating mainly from molds, to improvedigestibility of feed. In addition, there are several commerciallyavailable Saccharomyces cerevisiae yeast strains that when fed to cattlereportedly improve fiber digestion (Erasmus et al., (1992) J. Dairy Sci.75: 3056-3065; and Wohlt et al., (1998) J. Dairy Sci. 81: 1345-1352).Plant tissues and fibers can be disrupted to help release nutrients byadding organisms or enzymes produced by these organisms to forage beforefeeding or ensiling.

For example, in U.S. Pat. No. 6,037,161, an Aspergillus acetyl esterasenucleic acid sequence is described which encodes an enzyme with activitytowards acetylated xylans that may be used to modify plant materials forenhanced nutrient availability. However, these methods can be difficultand expensive to practice because recombinant technologies,fermentations, and chromatographic processes are required. In addition,the resultant enzyme has a narrow range of substrates and can fail toeffectively release nutrients from many plant materials.

Generally, for an animal to make efficient use of the feed it consumes,the energy demands of the microorganisms in the digestive tract must bemet and synchronized with the availability of plant proteins. A lack ofsynchrony will lead to a) proteins and other nutrients being poorlyutilized in the digestive tract, b) a loss of nitrogen, in urine andfeces and c) a need to feed excessive amounts of protein concentrates assupplements to the diet. The use of organisms and enzymes can improve orenhance the value of the feed animals receive and the performance of theanimals. For example, WO 92/10945 discloses such a combination for usein enhancing the value of prepared silage. WO 93/13786 and WO 96/17525relate to the enhancement of animal performance using microorganisms,while WO 93/13786 refers to a species of Lactobacillus.

Silage can be spoiled when aerobic organisms, such as some yeast andmolds, propagate in the stored feed. For example, silage can be damagedby air leaks in a silo or when air is introduced while farmers areremoving part of the silage. In the presence of oxygen, yeasts, molds,and aerobic bacteria can consume nutrients, and release unpleasant ortoxic metabolites. This elevated aerobic microbe activity has beenconsidered undesirable since it often results in aerobic spoilage ofsilage and the consequent depletion of silage dry matter (DM) nutrition.However, some microbes, such as killer yeast (see U.S. Pat. No.6,489,158) and Lactobacillus buchneri strains (see U.S. Pat. No.6,326,037) can beneficially inhibit growth of spoilage microbes therebystabilizing nutrient value of ensiled plant materials. A further methodto reduce this problem is to inoculate the silage with a fast growingmicrobe, such as Lactobacillus species, which release organic acids tolower the silage pH and inhibit growth of spoilage organisms.

SUMMARY OF INVENTION

It has now been found that acetyl esterase producing bacterial strainsor functional mutants thereof are suitable for use as a silage inoculantfor improving fiber digestibility.

Further it has been found that plant fiber digestion in an animal isenhanced by feeding the animal fibrous plant material treated with aneffective amount of an acetyl esterase containing composition, whereinthe acetyl esterase containing composition is derived from a acetylesterase producing bacterial strain or functional mutant thereof.

Embodiments of the present invention provide methods of treating animalfeed or silage with the acetyl esterase producing bacterial strainsdisclosed herein, as well as the treated animal feed or silage itself.Methods of improving animal performance by feeding the inoculated animalfeed or silage are also provided.

DETAILED DESCRIPTION OF INVENTION

Before describing the embodiments of the present invention in detail, itis to be understood that this invention is not limited to particularcompositions or methods of improving digestibility of ensiled forage,which can, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” can include plural referents unless thecontent clearly indicates otherwise. Thus, for example, reference to “acomponent” can include a combination of two or more components;reference to “feed” can include mixtures of feed, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which embodiments of the invention pertain. Many methods andmaterials similar, modified, or equivalent to those described herein canbe used in the practice of the embodiments of the present inventionwithout undue experimentation. In describing and claiming theembodiments of the present invention, the following terminology will beused in accordance with the definitions set out below.

The term “digestibility,” as used herein, refers to the ability toderive soluble nutrients from a feed plant material. Digestibility canbe determined, e.g., by analyses that provide assay data indicating theamount of feed residue remaining in a digestion and/or by analyses thatprovide assay data indicating the amount of nutrients released from feedin a digestion.

The term “nutrient availability,” as used herein, refers to the amountof soluble nutrients made available in a digestion. Nutrientavailability can be a measure of feed digestibility. Feed plant materialnutrient availability can be determined by assay of: feed plantmaterials, feed plant materials treated with compositions of theinvention, ensiled feed plant materials, in vitro digested feed plantmaterials, in situ digested feed plant materials, and/or the like.Assays for measurement of nutrient availability can include, e.g., gaschromatography, sugar assays, amino acid assays, free fatty acid assays,volatile fatty acid assays, carbohydrate assays, and/or the like.

The term “inoculation,” as used herein, refers to introduction of viablemicrobes to media or feed plant material.

The term “plant material,” as used herein, refers to material of plantorigin. Feed plant material is plant material intended to be fed to ananimal.

As used herein, “acetyl esterase” includes one or more enzymes thathydrolyze p-nitrophenyl acetate to form p-nitrophenol and acetic acid.Thus, the present invention contemplates the use of one or moreesterases with activity for p-nitrophenyl acetate as animal feedsupplements to improve the digestion of a high fiber diet. Enzymesclassified as acetyl esterases include, without limitation, xylan acetylesterases, mannan acetyl esterases and rhamnogalacturonan acetylesterases.

The term “conditioned media,” as used herein, refers to media of theembodiments of the invention in which acetyl esterase producingbacterial species have been grown. Such media are said to beconditioned, e.g., by the release of metabolites, inhibitors, and/orenzymes into the media from the acetyl esterase producing bacteria.

Units, prefixes, and symbols may be denoted in their SI accepted form.Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange.

As used herein, “functional mutant” means a bacterial strain directly orindirectly obtained by genetic modification of, or using, the referencedstrain(s) and retaining at least 50% of the activity of a control silageusing the referenced strain. The genetic modification can be achievedthrough any means, such as but not limited to, chemical mutagens,ionizing radiation, transposon-based mutagenesis, or via conjugation,transduction, or transformation using the referenced strains as eitherthe recipient or donor of genetic material.

As used herein, “isolated” means removed from a natural source such asfrom uninoculated silage or other plant material.

As used herein, “purified” means that a bacterial species or strain issubstantially separated from, and enriched relative to: yeasts, molds,and/or other bacterial species or strains found in the source from whichit was isolated.

As used herein, “animal performance” means the yield of meat, milk,eggs, offspring, or work.

The term “silage” as used herein is intended to include all types offermented agricultural products such as grass silage, alfalfa silage,wheat silage, legume silage, sunflower silage, barley silage, wholeplant corn silage (WPCS), sorghum silage, fermented grains and grassmixtures, etc.

As used herein, “pre-ensiled plant material” means grasses, maize,alfalfa and other legumes, wheat, sorghum, sunflower, barley andmixtures thereof. All of which can be treated successfully with theinoculants of the embodiments of the present invention. The inoculantsof the embodiments of the present invention are also useful in treatinghigh moisture corn (HMC).

An embodiment of the invention is a composition for use as a silageinoculant comprising an acetyl esterase producing bacterial strain or afunctional mutant thereof and a suitable carrier. Suitable acetylesterase producing bacterial strains or functional mutants thereofinclude Bacillus strains. Suitable acetyl esterase producing Bacillusstrains or functional mutants thereof include Bacillus subtilis andBacillus pumilus strains. Suitable acetyl esterase producing Bacillussubtilis and Bacillus pumilus strains include Bacillus subtilis, strainBS62, deposited as Patent Deposit No. NRRL B-30763, Bacillus subtilis,strain BS116, deposited as Patent Deposit No. NRRL B-30764, Bacillussubtilis, strain BS5482, deposited as Patent Deposit No. NRRL B-30765,Bacillus pumilus, strain BP5295, deposited as Patent Deposit No. NRRLB-30766, Bacillus pumilus, strain BP5579, deposited as Patent DepositNo. NRRL B-30767, Bacillus subtilis, strain BS5752, deposited as PatentDeposit No. NRRL B-30768, and mixtures thereof.

In an embodiment of the invention the composition contains from about10¹ to about 10¹⁰ viable organisms of the acetyl esterase producingbacterial strain or functional mutant thereof per gram of a pre-ensiledplant material. In a further embodiment of the invention the compositioncontains from about 10² to about 10⁷ viable organisms of the acetylesterase producing bacterial strain or functional mutant thereof pergram of a pre-ensiled plant material. In yet a further embodiment thecomposition contains from about 10³ to about 10⁶ viable organisms of theacetyl esterase producing bacterial strain or functional mutant thereofper gram of a pre-ensiled plant material.

Suitable carriers are either liquid or solid and are well known by thoseskilled in the art. For example, solid carriers may be made up ofcalcium carbonate, starch, cellulose and combinations thereof.

An embodiment of the invention is a biologically pure culture ofBacillus subtilis, strain BS62, deposited as Patent Deposit No. NRRLB-30763. A further embodiment of the invention is a biologically pureculture of Bacillus subtilis, strain BS116, deposited as Patent DepositNo. NRRL B-30764. Another embodiment of the invention is a biologicallypure culture of Bacillus subtilis, strain BS5482, deposited as PatentDeposit No. NRRL B-30765. An additional embodiment of the invention is abiologically pure culture of Bacillus pumilus, strain BP5295, depositedas Patent Deposit No. NRRL B-30766. A further embodiment of theinvention is a biologically pure culture of Bacillus pumilus, strainBP5579, deposited as Patent Deposit No. NRRL B-30767. Another embodimentof the invention is a biologically pure culture of Bacillus subtilis,strain BS5752, deposited as Patent Deposit No. NRRL B-30768.

A deposit of the following microorganisms was made on Sep. 1, 2004 withthe Agricultural Research Service (ARS) Culture Collection, housed inthe Microbial Genomics and Bioprocessing Research Unit of the NationalCenter for Agricultural Utilization Research (NCAUR), under the BudapestTreaty provisions. The strains were given the indicated accessionnumbers. The address of NCAUR is 1815N. University Street, Peoria, Ill.,61604. Bacillus subtilis, strain BS62, NRRL B-30763, Bacillus subtilis,strain BS116, NRRL B-30764, Bacillus subtilis, strain BS5482, NRRLB-30765, Bacillus pumilus, strain BP5295, NRRL B-30766, Bacilluspumilus, strain BP5579, NRRL B-30767, Bacillus subtilis, strain BS5752,NRRL B-30768. Applicant(s) will meet all the requirements of 37 C.F.R.§1.801-1.809, including providing an indication of the viability of thesample when the deposit is made. Each deposit will be maintained withoutrestriction in the ARS Depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theenforceable life of the patent, whichever is longer, and will bereplaced if it ever becomes nonviable during that period. The depositswill irrevocably and without restriction or condition be available tothe public upon issuance of a patent. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernment action.

A method for treating pre-ensiled plant material to enhance thedigestibility of the resulting silage by adding to the pre-ensiled plantmaterial a digestibility enhancing amount of a composition containing anacetyl esterase producing bacterial strain or a functional mutantthereof of is also disclosed. Suitable pre-ensiled plant materialsinclude grasses, maize, alfalfa and other legumes, wheat, sorghum,sunflower, barley and mixtures thereof.

An embodiment of the invention is a method for enhancing plant fiberdigestion in an animal by feeding a fibrous plant material treated withan effective amount of a acetyl esterase-containing composition to theanimal, wherein the acetyl esterase is derived from a acetyl esteraseproducing bacterial strain or a functional mutant thereof. Suitableacetyl esterase producing bacterial strains or functional mutantsthereof include Bacillus strains. Suitable Bacillus strains orfunctional mutants thereof include Bacillus subtilis and Bacilluspumilus strains. Suitable Bacillus subtilis and Bacillus pumilus strainsinclude Bacillus subtilis, strain BS62, deposited as Patent Deposit No.NRRL B-30763, Bacillus subtilis, strain BS116, deposited as PatentDeposit No. NRRL B-30764, Bacillus subtilis, strain BS5482, deposited asPatent Deposit No. NRRL B-30765, Bacillus pumilus, strain BP5295,deposited as Patent Deposit No. NRRL B-30766, Bacillus pumilus, strainBP5579, deposited as Patent Deposit No. NRRL B-30767, Bacillus subtilis,strain BS5752, deposited as Patent Deposit No. NRRL B-30768, andmixtures thereof.

The composition that is fed to the animal has been treated with aneffective catalytic amount of the acetyl esterase producing bacterialstrain or functional mutant thereof as is readily determinable by thoseskilled in the art in animal husbandry. Animals that are benefited byembodiments of the present invention are mammals and birds, includingbut not limited to ruminant, equine, bovine, porcine, caprine, ovine andavian species, e.g., poultry.

An embodiment of the invention is a substantially purified strain of abacterium selected from the group consisting of Bacillus subtilis,strain BS62, deposited as Patent Deposit No. NRRL B-30763, Bacillussubtilis, strain BS116, deposited as Patent Deposit No. NRRL B-30764,Bacillus subtilis, strain BS5482, deposited as Patent Deposit No. NRRLB-30765, Bacillus pumilus, strain BP5295, deposited as Patent DepositNo. NRRL B-30766, Bacillus pumilus, strain BP5579, deposited as PatentDeposit No. NRRL B-30767, Bacillus subtilis, strain BS5752, deposited asPatent Deposit No. NRRL B-30768, and mixtures thereof.

EXAMPLES

Embodiments of the present invention are further defined in thefollowing Examples. It should be understood that these Examples, whileindicating certain embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the embodiments of theinvention to adapt it to various usages and conditions. Thus, variousmodifications of the embodiments of the invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Testing of Bacillus Strains for Production of Acetyl EsteraseActivity

A total of about 5500 strains from one of the Microbial CultureCollections of Pioneer Hi-Bred International Inc., Des Moines, Iowa werescreened for acetyl esterase activity using 4-nitrophenyl acetate(pNP-acetate; Sigma Chemical Co., St. Louis Mo.) as the substrate.

A standard assay was used whenever a small number of strains were testedfor activity. For the standard assay, bacillus strains were streakedfrom freezer vials onto Tryptic soy agar (TSA, DIFCO, Becton, Dickinsonand Co., Sparks, Md.) and incubated at 30° C. for 72 h. Cultures wereinoculated directly from the TSA plates in to 10 mL of Tryptic soy broth(TSB) and grown at 30° C. for 24 h on a roller drum. Freshly preparedwhole plant corn broth (WPCB; 10 mL) was then inoculated with TSB growncultures and incubated at 30° C. for 48-72 h on a roller drum. WPCB wasprepared by suspending whole plant corn (WPC) (ground to pass a 1 mmscreen) in distilled (D) H₂O (3.4%; w/v), autoclaving (sterilizing) themixture (121° C., 15 min) and then cooling the mixture to roomtemperature prior to use. An uninoculated WPCB (control) treatment wasincluded at all times in order to verify that acetyl esterase activitywas the result of inoculation. Culture supernatants were collected bycentrifugation and freeze-dried or assayed immediately for acetylesterase activity as described below.

For the high throughput screening of about 5500 strains, strains wereinoculated into 96 well microtitre plates containing 200 μL of TSB perwell and incubated on a plate shaker at 150 rpm for 24 h at 30° C.Sterile WPCB was dispensed into a 96 deep well filtration microtiterplate (Millipore Cooperation, Billerica, Mass.) and each well was theninoculated with 20 μL of the 24 h TSB cultures using an 8-channelmicropipette. WPCB filtration microtiter plates were incubated at 30° C.for 72 h, with shaking at 150 rpm, and then filtered by vacuum using aMillipore vacuum manifold (Millipore Cooperation, Billerica, Mass.). Theculture filtrates were collected and assayed for acetyl esteraseactivity as described below.

Acetyl esterase activity was measured using a modification of theprocedures described by Huggins and Lapides (1947) (J. Biol. Chem. 170:467-482.) and Williams and Withers, (1981) (J. Appl. Bacteriol. 51375-385). Briefly, a standard stock solution of p-nitrophenol (10 mM, in0.05 M citrate phosphate buffer, pH 6.0) was prepared, dispensed into 1mL aliquots and these were kept frozen, and utilized individually whenrequired. The assays were performed in 96 well microtitre plate formatas shown in Table 1.

TABLE 1 Typical microtiter plate well assignments for acetyl esteraseactivity assay: ITEM Row and well numbers WPCB control¹ Row A wells 1-3.Buffer control² Row B wells 1-3. Standards #1-6³ Row C-H wells 1-3.Enzyme positive control⁴ Row H wells 10-12. BACILLUS TEST Remainingwells in triplicate SAMPLES ¹Whole plant corn broth ²Citrate phosphatebuffer (0.05 M; pH 6.0) ³See Table 2 for preparation of standards⁴Pectinex 3X L (Novozymes North America Inc., Franklinton. NC)A standard curve was prepared first by diluting a stock standard tenfold in 0.05 M (pH 6) citrate phosphate buffer (buffer) to produce aworking standard, and then by substituting working standard solutionwith buffer as shown in Table 2.

TABLE 2 Preparation of standard curve for acetyl esterase assay StandardWorking standard (μL) Buffer¹ 1 5 195 2 10 190 3 15 185 4 30 170 5 45155 6 60 140 ¹Citrate phosphate buffer (0.05 M; pH 6.0)

The final incubation volume in each well was 200 μL. Forty microlitersof buffer or WPCB were dispensed in to the microtitre plate well asillustrated in Table 1. Buffer, 160 μL, was then dispensed into thewells containing WPCB to account for background WPCB absorbance at 405nm.

Forty (40) microliters of a 1:500 dilution (in buffer) of Pectinex 3X L(Novozymes, North America Inc, Franklinton, N.C.) was dispensed asillustrated in Table 1. Pectinex 3X L was used as a positive control forthe acetyl esterase assay. Forty (40) microliters of culturesupernatants prepared as described above were then dispensed in themicrotitre plate wells as illustrated in Table 1. The substrate solutionwas prepared by dissolving 18.11 mg of 4-nitrophenyl acetate in 710 μLof dimethylsulphoxide and then this solution was diluted to 100 mL inbuffer. Using an 8-channel micropipette, 160 μL of substrate weredispensed into the buffer control wells and 160 μL of substrate weredispensed into wells 4-12 (see Table 1). A time zero absorbance reading(405 nm; Vmax Kinetic Microplate Reader, Molecular Devices, Menlo Park,Calif.) was taken immediately after addition of substrate and the platesthen incubated at 30° C.

The plates were read again after 30 min. and cultures that had acorrected absorbance of more that 0.09 (about 160 strains) were deemedto be acetyl esterase producers of interest. Under these assayconditions the actual range in absorbance (405 nm) for the producers ofinterest was 0.091 to 0.889. All bacillus strains incorporated in theproceeding examples belonged to this subset of about 160 acetyl esteraseproducing strains of interest.

For the freeze dried bacillus culture supernatants described in Examples2 and 3, freeze-dried material was diluted in de-ionized distilled waterand acetyl esterase activities were determined following a 60 min.incubation. For strains used as inoculants for silage (Bacillussubtilis, strain BS62, deposited as Patent Deposit No. NRRL B-30763,Bacillus subtilis, strain BS5482, deposited as Patent Deposit No. NRRLB-30765, Bacillus pumilus, strain BP5295, deposited as Patent DepositNo. NRRL B-30766, Bacillus pumilus, strain BP5579, deposited as PatentDeposit No. NRRL B-30767, Bacillus subtilis, strain BS5752, deposited asPatent Deposit No. NRRL B-30768) in Examples 4, 5 and 6, acetyl esteraseactivities were determined on supernatants harvested from 24 h TSBcultures. Concentrations of protein in the supernatant of these cultureswere then determined using the method of Bradford (Bradford, M, (1976)Anal. Biochem. 72 248-254). Specific acetyl esterase activities ofBP5295, BP5779, BS62, BS116, BS5482, and BS5752 were 94.6, 82.6, 28.0,45.2, 9.8 and 69.6 μmol pNP g protein⁻¹ min⁻¹, respectively.

Example 2 Effects of Culture Supernatants of Acetyl Esterase ProducingBacillus on Ruminal Digestibility of Corn SILAGE In Vitro

Proprietary Bacillus strains SB7.85 and X450 were selected for anexperiment to determine the effects of a crude enzyme rich WPCB culturesupernatant on digestibility of corn silage. Strains were streaked fromfreezer vials onto TSA (DIFCO, Becton, Dickinson and Co., Sparks, Md.)plates and incubated at 30° C. for 72 h. Cultures were then inoculateddirectly from the TSA plates in to 10 mL of TSB and grown at 30° C. for24 h.

Following incubation, 10 mL of TSB grown cultures was poured directlyinto 100 mL of freshly prepared WPCB. WPCB was prepared as described inExample 1. An additional treatment consisting of 100 mL of uninoculatedWPC broth was included as an uninoculated control and treated in exactlythe same way as the cultures. Inoculated and uninoculated WPCB wasincubated at 30° C. for 24 h and then transferred into ten 100 mL WPCBprepared as described above, and further incubated at 30° C. for 72 h.Supernatants were collected following centrifugation (26 000×g, 4° C.;30 min.), freeze-dried, and then assayed for acetyl esterase activity asdescribed in Example 1. Freeze dried supernatants were then stored inzip lock plastic bags at 4° C. Acetyl esterase activities of freezedried supernatants from X-450 and SB7.85 were 1.79 and 1.20 μmol pNP gsupernatant⁻¹ min⁻¹, respectively. WPCS was obtained from a bunker siloat the Pioneer Livestock Nutrition Center (PLNC), Sheldahl, Iowa, driedand ground to pass an 8 mm screen. For each treatment, WPCS (5 g) wasweighed into aluminum foil trays and spread evenly across the bottom ofthe trays. Fifty-five ((55), low), 110 (medium) and 550 (high) mg offreeze dried supernatants harvested from inoculated (SB7.8 and X-450)and uninoculated (control) broth cultures were re-suspended in 3 mL ofdistilled water in a 15 mL falcon tube, and mixed thoroughly to providea uniform solution. These solutions were then sprayed onto the silage,with regular and thorough mixing by use of a spatula, during and afterapplication. Treated silage was left at room temperature for 24 h andthen dried to constant weight in a forced air oven at 62° C.

For the determination of effects on in vitro ruminal fermentation, 5replicates of untreated (control) and treated silage (approximately 300mg) were accurately weighed into 100 mL serum vials (VWR Int., WestChester, Pa.). In vitro ruminal digestion studies were performed using amodification of the method of Pell and Schofield (1993) (J. Dairy Sci.vol 76 No 4, 1063-1073). Briefly, twenty-four (24) ml of the anaerobicincubation solution (phosphate-bicarbonate buffer and reducing solution;39° C.) of Goering, and Van Soest (Goering, H. K. and P. J. Van Soest(1970); Forage fiber analyses Handbook No. 379. ARS-USDA, Washington,D.C.), was added to the serum vials containing the silage under a streamof oxygen free CO₂. The serum vials were sealed with butyl septa(Geo-Microbial Technologies, Inc., Ochelata, Okla.) and then crimpedwith aluminum seals (VWR Int., West Chester, Pa.) and placed in a 39° C.incubator. Ruminal fluid obtained from two (2) steers fed a diet ofwhole plant corn silage (WPCS) for more than two (2) weeks prior toexperimentation, was strained through two (2) layers of cheese cloth,combined to create one solution and then maintained under a stream ofoxygen free CO₂. Strained rumen fluid, 6 mL, was then added to each ofthe serum vials at 39° C. under a stream of oxygen free CO₂ and themixtures incubated, with shaking (54 RPM) at 39° C. for 24 h. Blanksconsisting of serum vials with no silage but containing all the otherincubation mixture components were included as controls to account fornon-silage derived DM and volatile fatty acid (VFA).

On termination of the incubation, a 1 mL aliquot was collected from eachserum vial, processed and then analyzed for VFA concentrations using gaschromatography as is known in the art. The rest of the serum vialcontents were individually filtered through a pre-weighed piece ofnitrogen free nylon (approximately 10 cm by 10 cm; 40 microns + or − 15pore size; Ankom Technology Corp., Fairport, N.Y.) using a Buchner flaskattached to a vacuum source. The nylon pieces, now holding theincubation mixture residue for each sample, were folded over, dried to aconstant weight at 62° C., and then reweighed.

Dry matter disappearance (DMD) (%) was determined as the differencebetween the original silage weight and the weight of the residue fromthe serum vials, divided by the original silage weight and thenmultiplied by 100. The vials containing no silage were also filtered andweighed in the same manner in order to correct silage-containing vialsfor the DM arising from the rumen fluid only.

WPCS treated with supernatants prepared from SB7.85 and X-450 WPCBcultures had higher in vitro dry matter digestibility values thancontrol silages, which had been treated with supernatants fromuninoculated WPCB (Tables 3 and 4). For X-450, this effect was onlynoted at the middle application level (110 mg/5 g silage) but forSB7.85, all doses employed in the study increased DMD (Table 3).VFA concentrations produced from the WPCS treated with acetyl esterasecontaining bacillus supernatants were higher than the correspondingconcentrations derived from digestion of WPCS treated with uninoculatedsupernatants, reflecting the enhanced DMD values observed (Tables 3 and4).

TABLE 3 Effects of applying acetyl esterase containing Bacillus culturesupernatants to field grown WPCS on in vitro ruminal DMD (%), Gas (mL/gsilage) and VFA (mg/g) production from field grown WPCS. DMD Gas RuminalVFA produced (mg/g WPCS) Dose Treatment (%) (mL/g) Acetate PropionateButyrate Valerate Total VFA Low¹ Control 64.8 204 181  94.1 63.6 5.33344 X-450 63.1 179** 179  91.6 62.1 5.25 338 SB7.85 67.3* 211 182  98.764.5 6.68 352 Medium² Control 65.4 202 178  90.0 57.8 3.69 329 X-45067.8* 221** 181  97.4** 61.6 6.84* 346 SB7.85 67.1 224** 187* 100** 65.37.44* 360** High³ Control 63.6 214 176  90.5 56.4 5.05 328 X-450 64.3215 186* 102** 61.8 7.72 359** SB7.85 68.5** 227** 187* 106** 70.9**8.11* 372** SE⁴ 0.76  3.13  3.30  2.2 3.07 1.04  7.68 ¹55 mg freezedried supernatant/5 g whole plant corn silage. ²110 mg freeze driedsupernatant/5 g whole plant corn silage. ³550 mg freeze driedsupernatant/5 g whole plant corn silage. *, **significantly differentfrom control at P < 0.05 and P < 0.01, respectively. Means are leastsquare means. ⁴SE—standard error.

TABLE 4 Overall treatment effects of applying acetyl esterase containingBacillus culture supernatants to WPCS on in vitro ruminal DMD (%), Gas(mL/g silage) and VFA production (mg/g) from field grown WPCS at 48 h.DMD Gas Ruminal VFA produced (mg/g WPCS) Treatment (%) (mL/g) AcetatePropionate Butyrate Valerate Total VFA Control 64.6 207 179 91.4 59.24.69 334 X-450 65.1 205 182  96.9* 61.8* 6.60*  347* SB7.85 67.8** 221**  186** 102** 66.9** 7.41**  361** SE¹ 0.42 1.74 1.83  1.22 1.710.578 4.28 Trt x Dose ** ** NS² ** NS NS NS *, **Significantly differentfrom control at P < 0.05 and P < 0.01, respectively. Means are leastsquare means. ¹SE - standard error. ²NS - not significant.

As shown in the above Tables the application of acetyl esterasecontaining supernatants derived from Bacillus strains to ensiled WPCSenhances digestion of the treated WPCS.

Example 3 Effects of Culture Supernatants of Acetyl Esterase ProducingBacillus on Ruminal Digestibility of Corn Silage In Situ

Bacillus strains BP5295 (NRRL B-30766), BP5294, and proprietary strains5313 and SB7.85 were streaked from freezer vials on to TSA (DIFCO,Becton, Dickinson and Co., Sparks, Md.) plates and incubated at 30° C.for 72 h. Cultures were then inoculated as per the protocol given inExample 2 for the inoculation and growth of the Bacillus strains SB7.85and X450. Inoculated broths were then transferred by adding 5 mL ofculture into each of sixty 100 mL WPCB. Cultures were incubated at 30°C. for 72 h and the supernatants were collected following centrifugation(26 000×g, 4° C.; 30 min.), freeze-dried, and then assayed for acetylesterase activity as described in Example 1. Freeze dried supernatantswere then stored in zip lock plastic bags at 4° C. Acetyl esteraseactivities of freeze dried supernatants from BP5295, BP5294, 5313 andSB7.85 were 26.9, 9.4, 5.3 and 1.20 μmol pNP g supernatant⁻¹ min⁻¹,respectively.

WPCS was obtained from a bunker silo, dried and ground to pass an 8 mmscreen. WPCS (150 g) was weighed into each of twelve (12) large aluminumfoil trays and spread evenly across the bottom of the trays.

Freeze dried supernatants were re-suspended in 100 mL of distilled waterat three (3) concentrations (1.32, 5.23 and 10.56 g) and mixedthoroughly to provide a uniform solution. These solutions were thensprayed onto the silage, with regular and thorough mixing by use of aspatula, during and after application. Treated silage was left as atroom temperature for 24 h and then dried to constant weight in forcedair oven at 62° C.

Both micro (5.5 cm by 5.5 cm; 40 microns + or − 15; Ankom TechnologyCorp., Fairport, N.Y.) and macro in situ bags (20 cm by 24 cm; 40microns + or − 15; Ankom Technology Corp., Fairport, N.Y.) were used forthe ruminal determination of digestibility.

For the experiments performed using the micro in situ bags, DMD analysiswas concluded by incubating 0.5 g of dried ground silage in three (3)ruminally fistulated steers, which had been fed and adapted to a 100%WPCS diet for two (2) weeks prior to experimentation. For eachtreatment, two (2) repetitions were incubated in each of the 3-ruminallyfistulated steers for 24, 48 and 72 h.

For experiments carried out using the macro in situ bags, DMD wasdetermined as described above but only following a 48 h ruminalincubation of the medium level (5.23 g/150 g silage) only. Neutraldetergent fiber (NDF) and acid detergent fiber (ADF) concentrations ofthe silage before and after the 48 h ruminal incubation were determinedusing the Ankom Fiber Analyzer (Ankom Technology Corp., Fairport, N.Y.).

Original silage weights for each silage-containing micro in situ bagwere corrected for the A-fraction to give a corrected starting weight.The A-fraction was determined as the loss in silage DM when sealedsilage-containing micro in situ bags were immersed in water (15° C.) for15 min., washed gently for 2 min. on a slow cycle in a washing machineand then dried to constant weight at 62° C. Digestion coefficients forsilage NDF (NDFD %) and ADF (ADFD %) were determined as the differencein NDF or ADF concentration before and after ruminal incubation dividedby the concentration of NDF or ADF before ruminal incubation multipliedby 100.

WPCS treated with supernatants prepared from BP5294, BP5295, 5313 andSB7.85 WPC cultures had higher in vitro dry matter digestibility valuesthan control silages and these effects were most noted after 48 and 72 hof ruminal incubation, at the medium and high application levels ofapplication (Table 5).

TABLE 5 EFFECTS OF APPLYING ACETYL ESTERASE CONTAINING BACILLUS CULTURESUPERNATANTS TO WPCS ON IN SITU DMD (MICRO BAGS) Incubation Period (h)Treatment Dose 24 48 72 Control — 42.6 52.1 61.9 BP5294 Low¹ 44.4 57.963.3 Medium² 49.8 60.2* 68.1** High³ 44.8 59.4* 65.3* BP5295 Low 41.152.4 62.5 Medium 44.3 55.0 65.0 High 41.1 55.4 65.3 5313 Low 35.4 55.362.3 Medium 42.7 54.7 66.2* High 44.3 56.4 66.5* SB7.85 Low 42.2 57.665.1 Medium 45.7 58.6 64.1 High 47.2 58.4 67.4** SE⁴ 2.05 1.64 0.99¹1.32 g freeze-dried supernatant per 150 g whole plant corn silage ²5.23g freeze-dried supernatant per 150 g whole plant corn silage ³10.56 gfreeze-dried supernatant per 150 g whole plant corn silage ⁴SE, Standarderror. *, **Significantly different from control at P < 0.05 and P <0.01, respectively.Table 6 shows that NDFD levels were higher for WPCS treated with BP5294,BP5295 and 5313, and all treatments had higher levels of ADFD than theuntreated control.

TABLE 6 EFFECTS OF APPLYING ACETYL ESTERASE CONTAINING BACILLUS CULTURESUPERNATANTS TO WPCS (5.23 G/150 G) ON IN SITU DM, NDF AND ADFDISAPPEARANCE AT 48 H (MACRO BAGS) Digestion coefficients Treatment DMDNDFD ADFD Control 57.1 49.4 51.5 BP5294 61.6 52.2 57.6 BP5295 61.9 56.352.2 5313 61.3 52.5 56.7 SB7.85 59.1 49.4 53.3 SE 2.00 2.32 2.75

These results show that application of acetyl esterase containingsupernatants derived from bacillus strains BP5294, BP5295, SB7.85 and5313 improved in situ DM and fiber digestibility, and that this effectwas most pronounced after 48-72 h of ruminal incubation.

Example 4 Effects of Inoculation with Acetyl Esterase Producing Bacilluspumilus and B. subtilis Strains at Ensilage on In Vitro RuminalDigestion of Corn Silage

Green house (GH) grown corn plants, without tassels and ears wereobtained from Pioneer Hi-bred International, Inc. greenhouses inJohnston, Iowa. The plants were chopped using a Bearcat chipper/shredderand then blended with re-hydrated cracked corn kernels (3:2,respectively) to achieve a target DM of 350 g/kg. The cracked cornkernels were re-hydrated by mixing with H₂O (3:1, wt/vol, respectively)and leaving the mixture overnight at 4° C.

Acetyl esterase producing Bacillus pumilus strain BP5295 and Bacillussubtilis strains BS5752 and BS62 (see Example 1 for specific acetylesterase activities of these strains) were grown overnight TSB (DIFCO,Becton, Dickinson and Co., Sparks, Md.), on a roller drum, to reach acell count of about 1×10⁹ cfu/mL. For each strain, 0.45 mL of theovernight culture was added to 0.55 mL peptone-phosphate in a microfugetube to give 4.5×10⁸ cfu/mL. This 1 mL of culture was then applied to454 g of forage prepared as described above, to give an application rateof about 1×10⁶ cfu per gram. The treatments were applied to forage asaqueous solutions, by syringe dispersion, in a total of 2.2 mL/kg^(˜)1.The treatments were thoroughly mixed in to the forage by rolling theforage on a clean plastic sheet as they were applied to the forage.

Forage, 100 g, was ensiled, in quadruplicate for each treatment, inpolyethylene packet silos, which were vacuum packed and heat sealed asdescribed by Dennis et al. (1999) Page 87 In Proc. XII Int. Silage Conf.Swedish Univ. of Agric. Sci., Uppsala, Sweden.) using a Tilia FoodSaver, Professional II model (Tilia Inc. San Francisco, Calif.). Thepacket silos were incubated at room temperature, and after 45 d, thesilos were opened, emptied and the forage thoroughly mixed to give auniform mass. Aqueous extracts of silage were prepared by diluting ten(10) g in ninety-nine (99) mL of sterile distilled water and agitatingthe mixture in a stomacher (Stomacher 400, Seward limited, London,England) for 1 min. at the medium setting. Silage pH was determined onthese extracts immediately following preparation. The rest of thesilages were dried to constant at 62° C. weight and then ground to passan 8 mm screen.

For the determination of effects on in vitro ruminal fermentation,triplicate preparations of dried ground silage sample from each silo(approximately 300 mg) were accurately weighed into 100 mL serum vials(VWR Int., West Chester, Pa.). In vitro ruminal digestion studies wereperformed according to the protocols given in Example 2.

On termination of the incubation, a 1 mL aliquot was collected from eachserum vial, processed and then analyzed for VFA concentrations using theprotocols provided in Example 2.

DMD (%) was also determined according to the protocols provided inExample 2.

Bacillus pumilus strain 5295 and B. subtilis strains 5752 and 62 reducedsilage pH (P<0.05) indicating that the strains had grown and establishedin the silo (Table 7). Furthermore, BP5295 and BS5752 increased DMD(P<0.05) demonstrating that when used as silage inoculants, both B.subtilis and B. pumilus strains were able to enhance the digestibilityof corn silage. In vitro ruminal acetate, propionate and total VFAconcentrations produced were higher for BP5295 and BS5752 reflecting theincreases in DMD observed, although these latter differences were onlystatistically significant for BS5752 (P<0.05). Additionally both BP5295and BS5752 increased in vitro ruminal butyric acid production (P<0.05).

Inoculation with BS62 tended to increase silage digestion parameters,particularly VFA production; however, for DMD, these effects were not aspronounced as those observed for BS5752 and BP5295. While not wishing tobe bound by any one theory it is believed that not all Bacillus strainsthat produce acetyl esterase will necessarily improve digestibility ofensiled forage at all, or to the same extent, when used as inoculants.The degree to which successful establishment of the strain in the silois achieved will differ between strains. Tolerances to limitedconcentrations of oxygen, as well as the strain's competitive abilityunder ensiling conditions will play a significant role in determiningthe efficacy of selected strains.

TABLE 7 Effects of inoculating greenhouse grown corn with acetylesterase producing Bacillus strains on silage pH and in vitro ruminaldigestion of the resultant silage. mg produced per gram silage DM SilageDMD Acetic Propionic Isobutyric Butyric Isovaleric Valeric TotalTreatment¹ pH (%) Acid Acid Acid Acid Acid Acid VFA Control 3.83 63.2140 117 0.92 65.08 0.37 4.52 327 BP5295 3.78^(a) 65.7^(a) 143 123 1.0271.3^(a) 0.51 4.99 344 BS5752 3.78^(a) 68.4^(a) 150^(a) 128^(a) 1.0169.7^(a) 0.47 4.96 354^(a) BS62 3.78^(a) 64.5 145 123 1.09 68.4 0.324.59 343 SE² 0.013 0.51  2.46  2.25 0.061 1.09 0.168 0.23  5.12 ¹Foursilos were prepared for each treatment. ²SE, standard error.^(a)Significantly different from control at P < 0.05.

Example 5 Effects of Inoculation with Acetyl Esterase Producing Bacilluspumilus and B. subtilis Strains on Fermentation, Aerobic Stability andDigestibility of Silage Dry Matter and Fiber

Ryegrass was a first cut, harvested at the Pioneer Livestock NutritionCenter (PLNC), Sheldahl, Iowa in June 2004. The bacillus strains weregrown by the Quality Control and Microbial Production Group at PioneerHi-Bred in Johnston, Iowa, stabilized and lyophilized as is known in theart. The test strains were acetyl esterase producing (see Example 1 forspecific acetyl esterase activities of these strains) Bacillus pumilus5579 (BP5579), B. pumilus 5295 (BP5295), Bacillus subtilis 62 (BS62), B.subtilis 5752 (BS5752) and B. subtilis 5482 (BS5482). The ryegrass waseither uninoculated (control) or inoculated with the experimentalbacillus strains at 1×10⁶ colony forming units/gram forage (cfu/g). Thetreatments were applied to forage as aqueous solutions, by syringedispersion, and were thoroughly mixed into the forage by rolling theforage on a clean plastic sheet as the treatments were applied to theforage. For each treatment, four (4) experimental 4″×14″ polyvinylchloride (PVC) pipe silos were filled and packed at 100% packing density(approximately 230 kg DM M³), using a hydraulic press. Experimentalsilos were fitted with rubber quick caps at each end, held tight bymetal rings, and the top cap was equipped with a Bunsen valve to allowgasses to escape.

Silos were opened after 50 days of ensilage, emptied and the foragethoroughly mixed to give a uniform mass. Silage samples were allotted tothe various analyses, namely: pH, DM, Total N, ammonia N, Lactate andVFA and in situ rumen digestibility. Dry matter was determined by dryingto a constant weight in a forced air oven at 62° C. VFA, ammonia N andTotal N (crude protein/6.25) were determined by a commercial laboratory(Dairyland Laboratories, Arcadia, Wis.) using methods known in the art.Aerobic stability assessments were concluded on individual treatmentreplicates using the procedure of Honig (1986) (In Proc. Of the EurobacConf., P. Lingvall and S. Lindgren (ed.) 12-16 Aug. 1986. Swed. Univ. ofAgric. Sci. Grass and Forage Report No. 3-1990. Pp. 76-81. Uppsala,Sweden). The time (h) for silage temperature to rise 1.7° C. aboveambient was recorded (ROT). The integration of the area between theactual silage temperature curve and the line drawn by ambienttemperature (cumulative degree days or Cumm-DD) was calculated. Cumm-DDis a measure of total amount of heating.

Micro in situ bags (5.5 cm by 5.5 cm; 40 microns + or − 15; AnkomTechnology Corp., Fairport, N.Y.) were used for all ruminal incubations.In situ DMD analysis was concluded by incubating 0.5 g dried groundsilage in three (3) ruminally fistulated steers, which had been fed andadapted to a grass silage diet (95% grass silage and 5% dried rolledcorn on a dry matter basis) for two (2) weeks prior to experimentation.For each silo, one (1) repetition was incubated in each of the3-ruminally fistulated steers for 48 h; hence a total of 12 in situ bagswere incubated for each treatment. The A-fraction was determined as theloss in silage DM when sealed silage-containing micro in situ bags wereimmersed in water (15° C.) for 15 min., washed gently for 2 min. on aslow cycle in a washing machine and then dried to constant weight at 62°C.

NDF concentrations of the silage before and after the 48 h ruminalincubation were determined using the Ankom Fiber Analyzer (AnkomTechnology Corp., Fairport, N.Y.). Original silage weights for eachsilage-containing micro in situ bag were corrected for the A-fraction togive a corrected starting weight. Digestion coefficients for DM arepresented on both the original and the A-fraction-corrected startingweights. Digestion coefficients for silage NDF (NDFD %) were determinedas previously described.

Ryegrass silages were well fermented as illustrated by the terminal pHvalue of 4.08 for the control silages (Table 8). With the exception ofBS5482, all the bacillus strains reduced terminal silage pH (P<0.05) andin addition, tended to improve aerobic stability of silage, althoughthis latter effect was not statistically significant.

Interestingly, both BP5295 and BP5579 increased silage lactic acidconcentrations, with little effect on acetate whereas all three (3) B.subtilis strains (BS62, BS5482 and BS5752) did not influence lactic acidconcentrations but increased acetate concentrations.

Strains belonging to both species of Bacillus (B. pumilus and B.subtilis) increased dry matter digestibility (P<0.05). Furthermore bothspecies improved NDFD, with the most marked effects being noted forBP5295, BP5579 and BS62 (P<0.05).

Acetyl esterase producing Bacillus subtilis and B. pumilus strains grewand established in the silo as indicated by their ability to reducesilage pH. Furthermore, Bacillus strains from both species (B. pumilusand B. subtilis) improved DMD and NDFD of grass silage. These data showthat acetyl esterase producing B. pumilus and B. subtilis strainsimproved silage fermentation and dry matter and fiber digestibility whenused as silage inoculants.

TABLE 8 Fermentation, aerobic stability and in situ digestibility offirst cut ryegrass silages inoculated with Bacillus strains. ControlBP5295 BP5579 BS62 BS5482 BS5752 SE³ PH 4.08 4.01^(a) 4.03^(a) 4.05^(a)4.07 4.01^(a) 0.005 DM 34.5 31.8 32.2 33.6 33.2 31.2 0.77 Lactate (% DM)5.64 6.42^(a) 6.15^(a) 5.89 5.52 5.98 0.114 Acetate (% DM) 1.12 1.161.18 1.52^(a) 1.53^(a) 1.45^(a) 0.074 Total N (% DM) 2.20 2.45^(a)2.34^(a) 2.31^(a) 2.23 2.35^(a) 0.022 Ammonia N (% TN) 9.62 9.85 10.110.5 11.54 10.9 0.544 ROT (h) 12 49 102 86 12 86 31.7 CuMM-DD 2.67 1.9213.5 1.60 2.09 0.94 3.52 In situ DMD (% DM) 58.17 60.6^(a) 60.0^(a)60.4^(a) 60.4^(a) 61.0^(a) 0.40 In situ DMD-A-fraction² 41.1 44.3^(a)43.8^(a) 43.9^(a) 43.6^(a) 43.2 0.56 In situ NDFD 50.8 53.1^(a) 53.7^(a)53.3^(a) 51.3 51.7 0.68 ¹Four silos were prepared for each treatment.²Starting weight was corrected for the A-fraction. ³SE—standard error.^(a)Significantly different from control at P < 0.05.

Example 6 Effects of Inoculation with an Acetyl Esterase ProducingBacillus Strain Alone, and in Combination with a Lactobacillus plantarumStrain LP286 on Fermentation, Aerobic Stability and Digestibility ofSilage Dry Matter and Fiber

Ryegrass was a first cut, harvested at the Pioneer Livestock NutritionCenter (PLNC), Sheldahl, Iowa in June 2004. Acetyl esterase producingBacillus pumilus strain BP5579 (see Example 1 for specific acetylesterase activities of these strains) was grown by the Quality Controland Microbial Production Group at Pioneer Hi-Bred in Johnston, Iowa,stabilized and lyophilized as is known in the art. Similarly,Lactobacillus plantarum strain LP286 (ATCC# 53187) was grown by acontact manufacturer using procedures known in the art. The ryegrass waseither uninoculated (control) or inoculated with LP286 at 5×10⁴ colonyforming units/gram forage (cfu/g), BP5579 (1×10⁶ cfu/g) or BP5579/LP286(1×10⁶/5×10⁴ cfu/g). The treatments were applied to forage as aqueoussolutions, by syringe dispersion and thoroughly mixed in to the forageby rolling the forage on a clean plastic sheet as the treatments wereapplied to the forage.

For each treatment, four (4) experimental 4″×14″ polyvinyl chloride(PVC) pipe silos were filled and packed at 100% packing density(approximately 230 kg DM M³), using a hydraulic press. Experimentalsilos were fitted with rubber quick caps at each end, held tight bymetal rings, and the top cap was equipped with a Bunsen valve to allowgasses to escape.

Silos were opened after forty (40) days of ensilage, emptied and theforage thoroughly mixed to give a uniform mass. Silage samples wereallotted to the various analyses, namely: pH, DM, Total N, Ammonia N,Lactate and VFA and in situ rumen digestibility. Dry matter wasdetermined by drying to a constant weight in a forced air oven at 62° C.Aerobic stability assessments were concluded on individual treatmentreplicates using the procedure of Honig (1986) supra. The time (h) forsilage temperature to rise 1.7° C. above ambient was recorded (ROT). Theintegration of the area between the actual silage temperature curve andthe line drawn by ambient temperature (cumulative degree days orCumm-DD) was calculated. Cumm-DD is a measure of total amount ofheating.

Micro in situ bags (5.5 cm by 5.5 cm; 40 microns + or − 15; AnkomTechnology Corp., Fairport, N.Y.) were used for all ruminal incubations.In situ DMD analysis was conducted as described in Example 5. NDFconcentrations of the silage before and after the 48 h ruminalincubation were also determined as described in Example 5.

Ryegrass silages were well fermented as illustrated by the terminal pHvalue of 4.15 for the control silages (Table 9). All inoculanttreatments, including BP5579 alone reduced silage pH (P<0.05) andinoculation with BP5579 or BP5579/LP286 had little or no effect onaerobic stability of silage (ROT). Inoculation with BP5579 andBP5579/LP286 increased DMD and NDFD (P<0.05). The combination of BP5579and LP286 increased DMD and NDFD despite the fact the LP286 alonedecreased DMD and tended to decrease NDFD.

Acetyl esterase producing Bacillus pumilus strain BP 5579 grew andestablished in the silo as evidence by its ability to reduce silage pH.Furthermore, BP5579 improved DMD and NDFD of grass silage when usedalone or in combination with L. plantarum strain LP286. These datademonstrate that acetyl esterase producing B. pumilus is effective atimproving DMD and NDFD even when used in combination with traditionalsilage inoculant strains, which traditional silage inoculant strainswould normally be expected to dominate the Bacillus species duringsilage fermentation.

TABLE 9 PH, AEROBIC STABILITY AND DIGESTIBILITY OF FIRST CUT PRG SILAGESINOCULATED WITH BACILLUS STRAIN 5579, ALONE OR IN COMBINATION WITHLP286. BP5579/ Item¹ Control LP286 BP5579 LP286 SE³ PH 4.15 4.07^(a)4.04^(a) 4.02^(a) 0.008 DM 30.1 36.9^(a) 29.0^(a) 29.6 0.165 ROT (h) 9998 102 108 2.70 CuMM-DD 53 38.1 13.9 17.7 14.7 In situ DMD (% DM) 61.857.7 63.5^(a) 63.3^(a) 0.380 In situ DMD- 45.0 41.4 48.1^(a) 47.4^(a)0.54 A-fraction² In situ NDFD 46.5 44.9 50.9^(a) 50.6^(a) 0.87 ¹Foursilos were prepared for each treatment. ²Starting weight was correctedfor the A-fraction. ^(a)significantly different from control at P <0.05. ³SE - standard error.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

Having illustrated and described the principles of the embodiments ofthe present invention, it should be apparent to persons skilled in theart that the embodiments of the invention can be modified in arrangementand detail without departing from such principles. We claim allmodifications that are within the spirit and scope of the appendedclaims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or published patent document wasspecifically and individually indicated to be incorporated by reference.

1. A biologically pure strain of a bacterium selected from the groupconsisting of Bacillus subtilis, strain BS62, deposited as PatentDeposit No. NRRL B-30763, Bacillus subtilis, strain BS116, deposited asPatent Deposit No. NRRL B-30764, Bacillus subtilis, strain BS5482,deposited as Patent Deposit No. NRRL B-30765, Bacillus pumilus, strainBP5295, deposited as Patent Deposit No. NRRL B-30766, Bacillus pumilus,strain BP5579, deposited as Patent Deposit No. NRRL B-30767, Bacillussubtilis, strain BS5752, deposited as Patent Deposit No. NRRL B-30768,and mixtures thereof, wherein the bacterium enhances the digestibilityof silage when added to pre-ensiled plant material in digestibilityenhancing amounts.